1. Introduction

Cyclopeptidomimetics are analogs of natural occurring cyclopeptides, where original amide or disulfide bond was replaced with unnatural linker bridge. These kinds of compounds usually featured with unique restricted conformation, thereby making them to display enhanced metabolic stability and binding specificity to their molecular targets ^[1]. In addition, cyclization scaffold provides favorable benefits on other essential properties required for drugs, such as membrane permeability, bioavailability, and overall pharmacokinetics ^[2]. To date, cyclopeptidomimetics are dominantly used in treating infectious disease and oncology as well as in cardiovascular disease and immunology ^[3]. Therefore, many efforts have been made to explore efficient cyclization strategies for the synthesis of cyclopeptidomimetics.

Cyclopeptidomimetics usually prepared by solid phase peptide synthesis (SPPS) of linear precursors followed by in solution cyclization [1d, 3b, 4]. However, this cyclization step is still considered as a formidable challenge because intermolecular reaction proceeds much faster than intramolecular reaction, thus generating undesired linear oligomers and relevant cyclooligomers as by-products. As a result, time and cost consuming purification is needed to obtain high purity target compound for biological study. Using high dilution condition can only suppress this side reaction in limited success ^[5]. On the other hand, peptide on-resin cyclization strategy has emerged as a powerful approach for the synthesis of cyclopeptidomimetics, which perform the pivotal cyclization reaction while the linear peptide is still anchored on the resin. This strategy took advantage of the well-known “pseudodilution” effect by favoring intramolecular reaction over the intermolecular reaction ^[6]. And by-products could be easily separated by washing and filtration process. Moreover, on-resin cylcization can be utilized to construct combinatorial library by incorporation of multiple amino acids and perform post-cyclization modification for the hit-to-lead purpose in drug discovery. In this review, we focused on the recently developed on-resin peptide cyclization strategies for the efficient synthesis of cyclopeptidomimetics. The general methodologies of on-resin cylcization include: Cu (I)-catalyzed “click chemistry”, thio-ene “click chemistry”, ring-closing metathesis (RCM), intramolecular S_N2 or S_NAr nucleophilic substitute reaction and transition metal catalyzed intramolecular coupling reaction.

2. Syntheses of cyclopeptidomimetics 2.1. Cu(I)-catalyzed huisgen azide-alkyne cycloaddition (CuAAC)

Huisgen azide-alkyne cycloaddition (CuAAC), also defined as “click chemistry” by Barry Sharpless, is a cycloaddition reaction between an azide and an alkyne group under mild condition using copper as catalyst ^[7]. This reaction joined two species together by formation a five-membered 1, 2, 3-triazole ring linkage, which is believed to have similar physicochemical profiles as the amide bond ^[8]. In addition, the triazole unit is resistant to enzymatic degradation, and display excellent stability toward hydrolysis and oxidation. Furthermore, replacing amide bond with triazole linkage could generate interesting structures with unique conformation when binding with biological target.

Meldal’s group firstly reported on-resin CuAAC cyclization using PEGA (poly-(ethylene glycol)-poly(acrylamide)-copolymer) resin which have excellent swelling properties in various solvents ^[9]. PEGA amine resin was equipped with a base labile linker, HMBA (hydroxymethyl benzoic acid); then linear peptide Fmoc- Lys(Boc)-Dap(N₃)-His(Trt)-(D)Phe-Arg(Pmc)-Trp(Boc)-Pra-Met was assembled by standard Fmoc chemistry. Azide and alkyne modified amino acid, Fmoc-Dap(N₃)-OH and propargylglycine-OH, were incorporated into appropriate position, respectively, to give resin bound precursor 3 (Scheme 1). Linear peptide was detached from the resin using 0.1 mol/L NaOH and confirmed by HPLC. Both the side chain fully protected and deprotected peptide mimetics were used for the on-resin cyclization under typical click chemistry condition. On-resin cyclization proceeded smoothly in both cases to give cyclipeptidomimetics product 5 in 76% to 79% yield after work-up and purification by HPLC.

Finn’s group described the Wang-resin based peptide head-totail CuAAC cyclization under copper catalyst ^[10]. In their studies, 11-mer and 19-mer peptide 6 and 7 containing Arg-Gly-Asp (RGD) sequence were synthesized by standard Fmoc chemistry or Boc chemistry. Amino acids containing clickable function group, such as L-propargylglycines and 5-azidopentanoic acid, were introduced at the second position of C-terminal and N-terminal, respectively. Under “click chemistry” condition and subsequent cleavage from the solid support, cyclodimerization products 8 and 9 were generated selectively in 10%-20% of yield without detection of linear product after purification by HPLC and characterized by MALDI-TOF MS. Subsequent detailed mechanism study demonstrated that this unexpected cyclodimerization is independent of peptide sequence, but sensitive to the distance of alkyne group to resin and solvents composition. Cyclodimerization prefer to form α- and β-peptides but not δ-peptides (Scheme 2) ^[11].

Similarly, Khan’s group applied on-resin click chemistry to synthesize cyclic NGR and RGD peptides, which were further modified to give fluorescence labeled compound 14 ^[12]. The binding profile with aminopeptidase A, cell lysates from MCF-7 and SKOV-3 cancer cell lines was further investigated (Scheme 3). Recently, Zhang et al. utilized on-resin CuAAC strategy to synthesize two asymmetrical cyclopeptidomimetics CP1 and CP2, and further explored their self-assembly behavior in solution to better understand the mechanism of protein self-assembly ^[13].

Vidal and co-worker investigated the on-resin head-to-tail cyclization for the synthesis of vascular endothelial growth factor (VEGF) ligands using CuAAC reaction ^[14]. Based on the crystal structure data of protein, peptide mimics containing the essential amino acids are designed and synthesized by standard Fmoc chemistry on Rink amide MBHA resin. The L-propargylglycine was installed in the C-terminal and N-terminal azido glycine was introduced by either azido acid coupling or direct solid-phase diazo-transfer reaction using N-terminal amine group. The cyclization process was monitored by IR spectroscopy and modified Kaiser Test. Interestingly, cyclomonomeric products 17 are produced exclusively in their studies. Over all crude cyclization products are formed in a yield of around 50% and a purity of 70%. The structure was confirmed by ESI-mass and NMR spectroscopy after purification by RP-HPLC (Scheme 4). Later, Lokey’s group developed this strategy as a macrocyclization tool to prepare cyclic tetra-, penta-, hexa-, and heptapeptides successfully ^[15]. Reaction usually completed in 6 h at room temperature to give desired cyclopeptidomimetics 19 in 20%-75% isolated yield with small amount of cyclooligomers by-product (Scheme 5).amount of cyclooligomers by-product (Scheme 5).

Dawson’s group expanded on-resin click chemistry to side chain to side chain macrocyclization ^[16]. Full length MPER epitope of protein GP41 668-683 was chosen to demonstrate this side chain cyclization. Azido substitute lysine and L-propargylglycine were installed to the internal position of peptide sequence by standard Fmoc SPPS chemistry. The resin-bound peptide 20 was subjected to click chemistry condition, followed by deprotection and cleavage from the resin to afford 60%-75% side chain tethered peptides 21. Binding assay showed that this backbonemacrocyclization maintained significant affinity to protein 4E10 while relevant linear precursor displayed poor affinity. This side chain cyclization approach is useful for the optimization of peptides ligands and therapeutics in the future (Scheme 6).

2.2. Thiol-ene click chemistry

Thiol-ene chemistry is another type of “click chemistry”, where thiol and alkene undergo a radical addition to form a sulfide bond. Although this reaction is widely used in chemical biology and material chemistry ^[17], examples of using this strategy for the onresin synthesis of cyclopeptidomimetics are limited. As a proof of concept, Anseth’s group utilized this method to synthesize cycle RGD peptidomimetics ^[18]. Cysteine, alloc, or norbornene modified lysine were chosen as the function group, respectively. After linear peptide Ac-C(Mmt)RGDSfK(alkene) was built on the resin and Mmt protecting group was selectively removed, the thio-ene photocyclization reaction proceeded rapidly to give 23 in 24%-37% purification yield (Scheme 7). By replacing alkene group with alkyne, they further developed a sequential thiol-yne/thiol-ene photoreactions on-resin to prepare multivalent peptides 25 (Scheme 8) ^[19].

2.3. Ring-closing metathesis (RCM)

Ring-closing metathesis (RCM) is an intramolecular olefin metathesis reaction by the scission and regeneration of carbon- carbon double bonds catalyzed by Grubb’s catalyst ^[20]. This reaction has been proven to be a valuable tool in constructing cyclopeptidomimetics because the new generated ethylic bridge can significantly enhance the molecules chemical and metabolic stability, and improve bioactivity as well ^[21]. Lantibiotic is a type of biological active bacterial peptides with disulfide bridges. Synthesis of carbocyclic lantibiotic analogues could generate variants that are stable to oxidation and metabolism by replacing the fragile sulfide ring. Vederas’s group developed on-resin RCM strategy to synthesize ring-expanded analogues of lantibiotic peptides ^[22]. In this study, Fmoc-allyglycine was assembled by SPPS as RCM cyclization precursors, on-resin iterative RCM reaction catalyzed by Grubb’s catalyst generated carbocycle ring C, B, A sequentially; then SPPS chemistry was continued to introduce the left amino acid residue 10-15 to achieve complex lantibiotic analogs 30 (Scheme 9).

Verdine’s group explored on-resin RCM strategy to prepare a series of all-hydrocarbon crossed-linked cyclopeptides with ahelical structure ^[23]. In their synthesis, pentenylglycine residues were incorporated into the required position and followed by onresin RCM strategy to afford "-helical peptides 32. The properties of these peptides, such as chemical and thermal stability, protease resistance, target-binding affinity, and cell permeability, were greatly increased. These all-hydrocarbon cyclic peptides were successfully applied in number of biological systems (Scheme 10)

Bruke and co-worker designed several cyclo-polyproline helices which can bind with growth factor receptor bound protein 2 (Grb2) ^[24]. Amino acids and alkenyloxy modified prolines analogs were installed into the required position by SPPS synthesis to generate resin-bound RCM precursors 33. RCM reaction was performed on-resin using Grubb’s second generation catalyst, followed by cleavage and purification by HPLC to give cyclopeptidomimetics 34 (Scheme 11).

Amylin-(1-8) is a cyclic short peptide containing disulfide bond with promising therapeutic function in stimulating osteoblast proliferation. Brimble’s group applied RCM strategy to replace the unstable disulfide bond ^[25]. Allglycine or Alloclysine were incorporated into the linear peptide as replacement of cysteine/ 2 and cysteine/7 by SPPS. On-resin RCM reaction efficiency was optimized by increasing catalyst concentration when aiding with microwave heating. Solvents are important to this reaction as no reaction was observed when using dichorobenzene as solvent. High yield of RCM products was obtained after purification by HPLC. By using this protocol, 20 or 34 membered macrocycles 35-38 and 39 were prepared successfully (Scheme 12).

Dynorphin A is a linear natural occurring ligand for opioid receptors with low selectivity. To identify selective k opioid agonist, Aldrich’s group utilized RCM strategy to synthesize several cyclic ^{[2, 5]} and ^{[5, 8]} Dyn A analogues. The peptides were synthesized by SPPS and L/D-AllGly was incorporated into appropriate positions of linear precursor. After subject to 2nd generation of Grubb’s catalyst, deprotection, and cleavage, cyclization products were obtained in 56%-74% isolation yields. Both cis and trans isomers were generated in RCM reaction and could be separated by HPLC in a very slow gradient (0.1% increase per minute) elution. These Dyn A analogues displayed low nanomolar binding affinity to k opioid receptors (Scheme 13) ^[26].

2.4. Intramolecular S_N2 or S_NAr nucleophilic substitute reaction

S_N2 and S_NAr are two classic chemical transformations in organic synthesis, which are also demonstrated to be an effective strategy in constructing cyclopeptidomimetics on-resin. Burgess’s group applied on-resin cyclization S_NAr methodology to prepare βbturn cyclic peptidomimetics libraries ^[27]. Linear peptides were built on resin, 2-fluoro-5-nitrobenzoic acid and glycine derivatives containing NH₂, OH, SH was placed at the N- and C-terminal, respectively. After treatment with base and cleavage from the resin, a small library of cyclic peptidomimetics 43 was obtained in good purification yields (Scheme 14 and Scheme 15).

To screen and identify antibacterial reagents, Jefferson and coworker demonstrated that this methodology aiding with mixturebased combinatorial chemistry can be used to construct a cyclopeptidomimetics library 47 containing 12, 000 compounds (Scheme 16) ^[28]. Using similar method, they further built a cyclopeptidomimetics conjugates library having quinolone pharmacophore for antibiotics discovery ^[29].

In another investigation, Nefzi and co-worker described a parallel synthesis of thioether bridged cyclopeptidomimetics by using intramolecular S_NAr reaction ^[30]. The on resin cyclization occurred via the coupling of thio group with p-fluoro-mnitrobenzic acid. This straightforward approach produced a 19- membered macrocycles 52 in good yields and purities (Scheme 16).

On-resin intramolecular S_N2 displacement reaction is an alternative straightforward strategy for the synthesis of cyclopeptidomimetics. Burgess and co-worker applied this method to synthesizeβ-turn mimics, where thiol was used as nucleophile ^{[27, 31]}[27b, 31]. Head-to-tail cyclization was isolated as major product along with dimeric cyclization by-product (Scheme 17)

Nefzi et al. described parallel synthesis of 19-membered thioether cyclopeptidomimetics 56 via intramolecular thioether formation ^[32]. With the aid of microwave, the overall reaction efficiency and purity of crude products were greatly improved (Scheme 18).

Recently, Maayan and co-worker demonstrated that secondary amine at the N-terminus could serve as nucleophile toward alkyl halide under microwave irradiation ^[33]. They successfully applied this methodology to prepare a series of cyclopeptidomimetics including hydrophobic, chiral-helical, and metal-binding peptoids 58 (Scheme 19). The yields and purities were greatly enhanced as determined by HPLC. This method represents a practical tool to obtain functional cyclopeptidomimetics with wide application in medicinal chemistry.

2.5. Transition metal catalyzed intramolecular coupling reaction

Transition metal catalyzed cross-coupling reactions including Heck, Sonogashira, and Suzuki etc. are state-of-the-art methodologies in carbo-carbon bond formation chemistry, which has been widely applied in target and library diversification synthesis. Using this well-developed methodology in constructing cyclopeptidomimetics on resin has been investigated in recent years. Hauske and co-worker firstly demonstrated that intramolecular Heck coupling between acrylic acid and iodobenzene derivatives onresin can be used to generate macrocyclic molecules, where a small library 60 containing 20-24 members was prepared efficiently (Scheme 20) ^[34].

Likewise, Akaji, and co-worker used similar strategy and expanded this methodology to build complex macrocyclic peptidomimetics 63 containing RGD sequence with diverse side chain and varied ring size ^[35]. It is interesting to observed that reaction rate of macrocyclization on-resin is much faster than that in solution, which indicated that this methodology is especially suitable for solid support organic synthesis (Scheme 21). With the assistant of microwave energy, on-resin heck reaction completed rapidly in 1-30 min using modified procedure as reported by Byke et al. ^[36].

Spivey’s group is interested in design and synthesis of cyclopeptidomimetics containing rigid diphenylacetylene structure derived fromhuman IgE Cε3 domain ^[37]. The key macrocyclization step was constructed by intramolecular Sonogashira coupling. Resin anchored peptide derivative 64 was subjected to palladium and copper iodine catalyst, and head-to-tail cyclization proceeded successfully to give product 66 in 15% yield after cleavage and purification. It should be noted that resin type play vital role in this coupling, where cyclization only observed when using Rink resin but not NovasynTGR resin (Scheme 22).

Recently, on-resin intramolecular Suzuki coupling was exploited as an efficient strategy for the synthesis of biaryl bridged cyclopeptidomimetics. Planas et al. firstly prepared resin bound linear peptide precursor 67 containing both the boronate and the halogenated derivatives of aromatic amino acids ^[38]. Upon treatment with palladium catalyst and ligand under microwave irradiation at 120 ℃ for 30 min, the expected cyclic biaryl peptidomimetics 68 with varied ring size were obtained in 72%- 79% purity and in 23%-35% yield after purification. It is worth to note that boronate is unstable in Fmoc removal step resulting in hydrolysis to phenolic compound. Therefore the introduction of boronated aromatic amino acid should be placed at the last step. They further extended this methodology to synthesize biaryl cyclopeptidomimetics containing 3-aryltyrosine, which is a unique structure presenting in some of nature occurring biaryl cyclic peptides (Scheme 23) ^[39].

3. Conclusion and outlook

Many strategies have been developed to synthesize cyclopeptidomimetics on-resin recently. By using the well-known “pseudodilution” effect, the undesired side-product is significantly reduced and cyclization product is produced selectively. Thus, target product can be isolated efficiently in high purity for next biological study. The power of on-resin cyclization combined with combinatorial chemistry technology has shown great potential in generating large amount of compounds for high throughput screening, hit identification, and lead optimization in drug discovery. However, compared with the advance of modern organic synthetic methodology, application of these latest developments for on-resin cyclization to synthesize cyclopeptidomimetics is still limited. It is thus expected that new and more application of on-resin cyclization using